Monoclonal antibody DS6, tumor-associated antigen CA6, and methods of use thereof

ABSTRACT

The present application describes a monoclonal antibody selected from the group consisting of monoclonal antibody DS6, monoclonal antibodies that specifically bind to the antigen or epitope bound by monoclonal antibody DS6, and fragments of the foregoing that specifically bind to the antigen or epitope bound by monoclonal antibody DS6. Methods of use of such antibodies and the isolated antigen bound by such antibodies are also described.

RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 09/641,499, filed Aug. 18, 2000, the disclosure ofwhich is to be incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention concerns the monoclonal antibody DS6,tumor-associated antigen CA6, and methods of use thereof.

BACKGROUND OF THE INVENTION

[0003] Malignancy-associated changes of tumor cell membranesialoglycoproteins and sialoglycolipids (gangliosides) have beenobserved for decades and are implicated in many aspects of thetransformed phenotype, including altered adhesive properties andmetastatic potential (Bhavanandan, V. P., Furukawa, K., In: A. Rosenberg(ed.), Biology of the Sialic Acids, pp. 144-196, Plenum Press, New York(1995)). Altered, re-expressed or over-expressed sialoglycoconjugates,viewed as tumor-associated antigens (TAAs), are being investigated astargets for a variety of antibody-directed cancer therapies. Someexamples include monoclonal antibodies (mabs) directed against sTn(TAG-72) in breast cancer therapy (Estava, F. J. and Hayes, D. F.Monoclonal antibody-based therapy of breast cancer. In: M. L. Grossbard(ed.), Monoclonal antibody-based therapy of cancer, pp. 309-338, MarcelDekker, New York (1998)) and mabs to gangliosides such as GD2, GD3 andGM2 in the therapy of lung cancer and melanoma (Butler, M. O. andHaluska, F. G., In: M. L. Grossbard (ed.), Monoclonal antibody-basedtherapy of cancer, pp. 339-36⁴, Marcel Dekker, New York (1998); Grant,S. C. et al., In: M. L. Grossbard (ed.), Monoclonal antibody-basedtherapy of cancer, pp. 365-395, Marcel Dekker, New York (1998)).Nevertheless, there remains a need for new ways to detect and treatcancer in patients.

SUMMARY OF THE INVENTION

[0004] The present invention is based upon the development of murinemonoclonal antibody DS6. This antibody immunohistochemically reacts withan antigen, CA6, that is expressed by human serous ovarian carcinomasbut not expressed by normal ovarian surface epithelium or mesothelium.The CA6 antigen has a limited distribution in normal adult tissues andis most characteristically detected in fallopian tube epithelium, innerurothelium and type 2 pneumocytes. Pretreatment of tissue sections witheither periodic acid or neuraminidase from Vibrio cholerae abolishesimmunoreactivity with DS6 indicating that CA6 is aneuraminidase-sensitive and periodic acid-sensitive sialic acidglycoconjugate (“sialoglycotope”). SDS-polyacrylamide gelelectrophoresis of OVCAR5 cell lysates reveals the DS6 epitope to beexpressed on an 80 kiloDalton nondisulfide-linked glycoproteincontaining N-linked oligosaccharides. Two-dimensional nonequilibrium pHgradient electrophoresis gels indicates an isoelectric point ofapproximately 6.2-6.5. DS6 immunostaining can be partially diminished bypretreatment of tissue sections with chloroform/methanol, suggestingthat DS6 may also be expressed as a glycolipid. Comparison of theimmunohistochemical distribution of the CA6 antigen in human serousovarian adenocarcinomas reveals similarities to that of CA125, however,distinct differences and some complementarity of antigen expression arerevealed by double-label, two-color immunohistochemical studies. TheDS6-detected CA6 antigen appears distinct from other well-characterizedtumor-associated antigens, including MUC1, CA125, and the histo-bloodgroup-related antigens, sLea, sLex and sTn.

[0005] A first aspect of the present invention is a monoclonal antibodyselected from the group consisting of monoclonal antibody DS6 andmonoclonal antibodies that specifically bind to the antigen or epitopebound by monoclonal antibody DS6, and fragments of the foregoing thatspecifically bind to the antigen or epitope bound by monoclonal antibodyDS6.

[0006] A second aspect of the present invention is a method of screeningfor the presence of cancer in a human subject, comprising the steps of:(a) contacting a biological sample taken from said subject with anantibody as described above under conditions permitting said antibody tospecifically bind an antigen in the sample to form an antibody-antigencomplex; and then (b) determining the amount of antibody-antigen complexin the sample as a measure of the amount of antigen in the sample,wherein an elevated level of the antigen in the sample is associatedwith the presence of cancer in said subject.

[0007] A further aspect of the present invention is a method of treatingcancer in a subject in need thereof, comprising, administering to asubject afflicted with cancer a monoclonal antibody as described abovein a therapeutically effective amount.

[0008] A still further aspect of the present invention is isolatedtumor-associated antigen CA6, an about 80 kDa N-linked glycoprotein,reduced or non-reduced, with a PI of about 6.2-6.5, and containing asialiadase and periodate sensistive epitope called DS6. Such antigen maybe isolated by affinity purification with an monoclonal antibody DS6 asdescribed herein.

[0009] The foregoing and other objects and aspects of the presentinvention are explained in detail in the drawings herein and thespecification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1: DS6 immunohistochemical staining pattern on normal humantissues, A-D, with AEC (red) as the chromagen, hematoxylin counterstain:(A) Fallopian tube with apical staining of cells lining lumen, (B)apical aspect of type 2 pneumocytes of lung, (C) transitional epitheliumof ureter with DS6 staining of luminal-facing aspects of inner celllayers, (D) squamous metaplasia of uterine cervix with rim patternoutlining cell membrane.

[0011]FIG. 2: (A) DS6 immunoperoxidase buffer control, ie, withoutneuraminidase, on serous ovarian carcinoma, (B) DS6 immunoperoxidase onserous ovarian carcinoma following neuraminidase (Vibrio cholerae)treatment. C-F: Double label, sequential two color immunohistochamicalstaining of serous ovarian carcinomas with DS6 and OC125, hematoxylincounterstain: (C) DS6 (DAB, brown) followed by mouse Ig control (VIP,purple) shows a typical staining pattern for DS6 on tumors with bothluminal and cytoplasmic staining, (D) OC125 (DAB, brown) followed bymouse Ig control (VIP, purple) shows OC125 with a luminal pattern and afocus demonstrating a rim pattern along tumor cell membranes, (E) DS6(DAB) staining followed by OC125 (VIP) on a tumor demonstrating adiscreet area of staining (purple chromagen) that is DS6-nonreactive butOC125-reactive, (F) OC125 (DAB) staining followed by DS6 (VIP) stainingreveal areas of DS6-reactivity that were not detected by OC125.

[0012]FIG. 3: (A) Two-dimensional nonequillibrium pH gradient gelelectrophoresis (NEPHGE) analysis of the DS6-detected CA6 antigen.Samples were analyzed on NEPHGE gels (pI range 3.2-7.8) in the firstdimension, followed by 13% SDS-PAGE gels in the second dimension, allunder reducing conditions. (B) One dimensional SDS-PAGE analysis on 13%gels under reducing conditions and in (C) on 10% gels under nonreducingconditions.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Any type of antibody may be used in the present invention. Theterm “antibodies” as used herein refers to all types of immunoglobulins,including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG areparticularly preferred. The antibodies may be monoclonal or polyclonal(with monoclonal antibodies preferred) and may be of any species oforigin, including (for example) mouse, rat, rabbit, horse, or human.See, e.g., M. Walker et al., Molec. Immunol. 26, 403-11 (1989). Antibodyfragments that retain specific binding to the protein or epitope boundby DS6 are included within the scope of the term “antibody” and include,for example, Fab, F(ab′)2, and Fc fragments, and the correspondingfragments obtained from antibodies other than IgG. Such fragments can beproduced by known techniques. The antibodies may be chimeric orhumanized, particularly when they are used for therapeutic purposes.

[0014] Subjects or patients with which the instant invention isconcerned are primarily human subjects, but the invention may also beemployed with other mammalian subjects such as dogs, cats, and horsesfor veterinary purposes. Subjects may be male or female.

[0015] Applicants specifically intend that all United States patentreferences cited herein be incorporated herein by reference in theirentirety.

[0016] 1. Antibody Production.

[0017] Monoclonal antibodies of the present invention may be preparedusing any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include, but arenot limited to, the hybridoma technique, the human B-cell hybridomatechnique, and the EBV-hybridoma technique (Kohler, G. et al. (1975)Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030;Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120). Briefly, theprocedure is as follows: an animal is immunized with antigen orimmunogenic fragments or conjugates thereof. For example, haptenicoligopeptides of antigen can be conjugated to a carrier protein to beused as an immunogen. Lymphoid cells (e.g. splenic lymphocytes) are thenobtained from the immunized animal and fused with immortalizing cells(e.g. myeloma or heteromyeloma) to produce hybrid cells. The hybridcells are screened to identify those which produce the desired antibody.

[0018] Human hybridomas which secrete human antibody can be produced bythe Kohler and Milstein technique. Although human antibodies areespecially preferred for treatment of human, in general, the generationof stable human-human hybridomas for long-term production of humanmonoclonal antibody can be difficult. Hybridoma production in rodents,especially mouse, is a very well established procedure and thus, stablemurine hybridomas provide an unlimited source of antibody of selectcharacteristics. As an alternative to human antibodies, the mouseantibodies can be converted to chimeric murine/human antibodies bygenetic engineering techniques. See V. T. Oi et al., Bio Techniques4(4):214-221 (1986); L. K. Sun et al., Hybricloma 5 (1986).

[0019] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (S. L. Morrison, et al. Proc. Natl.Acad. Sci. 81, 6851-6855 (1984); M. S. Neuberger et al., Nature312:604-608 (1984); S. Takeda, S. et al., Nature 314:452-454 (1985)).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceCA6-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (D. R.Burton, Proc. Natl. Acad. Sci. 88,1120-3 (1991)).

[0020] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature (R. Orlandi et al., Proc. Natl. Acad. Sci. 86, 3833-3837(1989)); G. Winter et al., Nature 349, 293-299 (1991)).

[0021] Polyclonal antibodies used to carry out the present invention maybe produced by immunizing a suitable animal (e.g., rabbit, goat, etc.)with the antigen to which monoclonal antibody DS6 binds, collectingimmune serum from the animal, and separating the polyclonal antibodiesfrom the immune serum, in accordance with known procedures. Depending onthe host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gets such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Amongadjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0022] Antibodies that bind to the same epitope (i.e., the specificbinding site) that is bound by the DS6 antibody can be identified inaccordance with known techniques, such as their ability to compete withlabeled antibody to in binding to CA6 in a competitive binding assay.

[0023] Monoclonal antibodies specific for CA6 epitope can be used toproduce anti-idiotypic (paratope-specific) antibodies. See e.g.,McNamara et al., Science 220,1325-26 (1984), R. C. Kennedy, et al.,Science 232,220 (1986). These antibodies resemble the CA6 epitope andthus can be used as an antigen to stimulate an immune response againstCA6, or to screen other antibodies for the ability to specifically bindto the same epitope bound by monoclonal antibody DS6.

[0024] DS6 can also be bound to a column (such as Protein A/G) and usedto obtain purified CA6 antigen from a variety of sources, includinghuman tissues/tumors and cancer cell lines that produce CA6. Suchpurified CA6 antigen can then be used to produce additional antibodies(monoclonal and/or polyclonal) by methods described above. Some of theseantibodies may react with the DS6 epitope while others can recognizedifferent epitopes on CA6. In one example, enzyme immunoassay to detectantigens in human body fluids often use a combination of antibodies thatrecognize different, non-sterically interfering epitopes on the sameantigen. For this purpose, a column containing immobilized neuraminidasecould be used to desialylate purified CA6; the desialylated CA6 can thenbe used as an immunogen to produce antibodies (monoclonal and/orpolyclonal) that react with non-DS6 epitopes on CA6. These antibodiescould then be used as either capture and/or tracer antibodies in anenzyme immunoassay for quantitation of the CA6 antigen for use inmonitoring of CA6 in pathologic states.

[0025] 2. Immunoassay Techniques.

[0026] Those skilled in the art will be familiar with numerous specificimmunoassay formats and variations thereof which may be useful forcarrying out the method disclosed herein. See generally E. Maggio,Enzyme-Immunoassay, (1980)(CRC Press, Inc., Boca Raton, Fla.); see alsoU.S. Pat. No. 4,727,022 to Skold et al. titled “Methods for ModulatingLigand-Receptor interactions and their Application,” U.S. Pat. No.4,659,678 to Forrest et al. titled “Immunoassay of Antigens,” U.S. Pat.No. 4,376,110 to David et al., titled “Immunometric Assays UsingMonoclonal Antibodies,” U.S. Pat. No. 4,275,149 to Litman et al., titled“Macromolecular Environment Control in Specific Receptor Assays,” U.S.Pat. No. 4,233,402 to Maggio et al., titled “Reagents and MethodEmploying Channeling,” and U.S. Pat. No. 4,230,767 to Boguslaski et al.,titled “Heterogenous Specific Binding Assay Employing a Coenzyme asLabel.”

[0027] Antibodies as described herein may be coupled or conjugated to asolid support suitable for a diagnostic assay (e.g., beads, plates,slides or wells formed from materials such as latex or polystyrene) inaccordance with known techniques, such as precipitation. Antibodies asdescribed herein may likewise be conjugated to detectable groups such asradiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradishperoxidase, alkaline phosphatase), fluorescent labels (e.g.,fluorescein), chemiluminescent labels (e.g., acridinium groups,metalloporphyrins such as phthalocyanine dyes, luminol, etc.), metalatoms (e.g., technetium-99m), etc., in accordance with known techniques.See, e.g., U.S. Pat. No. 4,472,509 to Gansow (metal chelates tomonoclonal antibodies); U.S. Pat. No. 5,061,641 to Schochat et al.; andU.S. Pat. No. 4,861,869 to Nicoleotti et al. (radiolabelling proteins).

[0028] Immunoassays, or other types of assays to detect and/orquantitate the level of the CA6 antigen in samples as described below,may be used in screening assays to detect pathologic states associatedwith aberrant levels of CA6 expression (e.g., tumors, inflammatorystates), diagnostic studies, prognostic studies, or to monitor theprogression or diminution of CA6 expression in correlation with diseasestate.

[0029] Samples that may be collected for use in carrying out theimmunoassay may be tissue samples from the organ or tissue of interestwithin the subject, such tissue generally of most interest being thosetypes of tissues/cells that express differing amounts of CA6 inpathologic states as compared to non-pathologic states, or biologicalfluids such as blood (including blood fractions such as blood plasma orblood serum), urine, cerebrospinal fluid, etc). Examples may includeoverexpression or aberrant expression of CA6 in various types ofmalignancies as will be seen in the Tables below (e.g ovarian cancer,endometrial cancer, pancreatic cancer, breast cancer, urinary bladdercancer, lung cancer, etc.), as well as overexpression or aberrantexpression in other pathologic states, such as overexpression of CA6 bypneumocytes in lungs disease, for example, pneumonia.

[0030] A biological sample may be a cell sample, with an interveningculturing step being performed between the time the cell sample iscollected from the subject and the immunoassay is carried out on thebiological sample.

[0031] For immunohistological techniques, a tissue sample is collectedfrom the subject, and the presence or absence of binding of an antibodyof the invention is detected. The presence of binding of the antibody inan abnormal pattern or a pattern indicative of a tumor or cancerindicates the presence of a tumor or cancer in the subject from whichthe tissue sample is collected. The presence of the antigen in ametastatic tumor deposit can also be used to determine a likely sourceof the primary tumor. Any suitable immunohistology format may be used.The tissue sample may include patient biopsies, resections or cells forcytologic study. A similar technique to immumohistogy is the use ofsimilar techniques to detect and/or phenotype cells in body fluids orother suspensions as is used for flow cytometric examination.

[0032] For in vivo diagnostic purposes the antibody according to theinvention is coupled to or provided with a suitable externallydetectable label, such as e.g. a radiolabel as described above or ametal atom (e.g., technetium-99m), and administered to a subject (e.g.,by intraveneous or intraarterial injection), in an amount sufficient toproduce an externally detectable signal, whereupon the possiblelocalized accumulation of antibody in the body is determined, with alocalized accumulation of the antibody (in a region other than thatwhich would ordinarily be expected for normal subjects or subjects freeof disease) indicating the present of a tumor in that subject.

[0033] 3. Therapeutic Antibodies and Methods.

[0034] Monoclonal antibodies used for therapy (i.e., in a method ofcombatting cancer) may be monoclonal antibodies per se or monoclonalantibodies coupled to a therapeutic agent. Such antibodies are referredto herein as therapeutic monoclonal antibodies. Any therapeutic agentconventionally coupled to a monoclonal antibody may be employed,including (but not limited to) radioisotopes, cytotoxic agents, andchemotherapeutic agents (See generally Monoclonal Antibodies and CancerTherapy (R. Reisfeld and S. Sell Eds. 1985)(Alan R. Liss Inc. NY); U.S.Pat. No. 5,558,852 to Bigner and Zalutsky; U.S. Pat. No. 5,624,659 toBigner and Zalutsky).

[0035] Therapeutic agents may be conjugated or coupled to the antibodyby direct means or indirect means (e.g., via a chelator), such as thelodogen method or with N-succinimidyl-3-(tri-n-butylstanyl)benzoate (the“ATE method”), as will be apparent to those skilled in the art. See,e.g., M. Zalutsky and A. Narula, Appl. Radiat. Isot. 38, 1051 (1987).

[0036] Examples of radioisotopes which may be coupled to a therapeuticmonoclonal antibody include, but are not limited to, ¹³¹I, ⁹⁰Y, 211At,²¹²Bi, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re, and ²¹²Pb. Examples of chemotherapeuticagents which may be coupled to a therapeutic monoclonal antibodyinclude, but are not limited to, methotrexate. Examples of cytotoxicagents which may be coupled to a therapeutic monoclonal antibodyinclude, but are not limited to, ricin (or more particularly the ricin Achain).

[0037] The monoclonal antibodies of the invention can be conjugated toand used as targeting agents for genes (immunogenes, suicide genes),immunoliposomes, boron neutron capture therapy, photosensitizers forphotodynamic therapy, and other types of therapies that can be directedby antibodies.

[0038] It will be appreciated that monoclonal antibodies per se whichare used as therapeutic monoclonal antibodies incorporate those portionsof the constant region of an antibody necessary to evoke atherapeutically useful immunological response in the subject beingtreated.

[0039] Therapeutic monoclonal antibodies may be provided in lyophylizedform in a sterile aseptic container or may be provided in apharmaceutical formulation in combination with a pharmaceuticallyacceptable carrier, such as sterile pyrogen-free water or sterilepyrogen-free physiological saline solution.

[0040] The therapeutic methods disclosed herein may be employed withsubjects suspected of having a variety of tumors, whether primary ormetastatic or micrometastatic (see Tables below), of particularimportance are tumors of the ovary, endometrium, breast, urinarybladder, pancreas and lung. DS6 may also be of therapeutic use in othertypes of neoplasms, especially if used as part of a panel or combinationof therapeutic antibodies, each with different specificities (Smith, N.L. et al, Human Antibodies, 9, 61-65, (1999); Oldham, R. K., MolBiother., 3, 148-161, (1991)).

[0041] For administration, the antibody will generally be mixed, priorto administration, with a non-toxic, pharmaceutically acceptable carriersubstance (e.g. normal saline or phosphate-buffered saline), and may beadministered using any medically appropriate procedure, e.g.,intravenous or intra-arterial administration, injection into thecerebrospinal fluid). In certain cases, intradermal, intracavity,intrathecal or direct administration to the tumor or to an arterysupplying the tumor is advantageous.

[0042] Dosage of the antibody will depend, among other things, on thetumor being treated, the route of administration, the nature of thetherapeutic agent employed, and the sensitivity of the tumor to theparticular therapeutic agent. For example, the dosage will typically beabout 1 to 10 micrograms per Kilogram subject body weight. In anotherexample, where the therapeutic agent is ¹³¹I, the dosage to the patientwill typically be from 10 mCi to 100, 300 or even 500 mCi. Statedotherwise, where the therapeutic agent is 131I, the dosage to thepatient will typically be from 5,000 Rads to 100,000 Rads (preferably atleast 13,000 Rads, or even at least 50,000 Rads). Doses for otherradionuclides are typically selected so that the tumoricidal dose willbe equivalent to the foregoing range for ¹³¹I. The antibody can beadministered to the subject in a series of more than one administration,and regular periodic administration will sometimes be required.

[0043] The present invention is explained in greater detail in thefollowing non-limiting Examples. Abbreviations used: mab (monoclonalantibody), TAA (tumor-associated antigen), IH(immunohistochemistry/immunohistochemical), kiloDalton (kDa).

EXAMPLE 1

[0044] Monoclonal Antibody DS6 Detects a Tumor-Associated SialoglycotopeExpressed on Human Serous Ovarian Carcinomas

[0045] We have recently developed a murine monoclonal antibody (mab),clone DS6, using human serous ovarian carcinoma as the immunogen (Smith,N. L. et al., Human Antibodies, 9, 61-65, (1999); Kearse, K. P. et al,Int. J. Cancer, in press, (2000)). We now report the immunohistochemical(IH) distribution of the DS6-detected antigen, CA6, in normal adulthuman tissues and selected neoplasms and characterize the epitope as asialoglycotope.

[0046] 1. Materials and Methods

[0047] Hybridoma production. Immunizations, fusions and screening wereperformed essentially as described previously using P3×63-Ag8.653myeloma cells with human serous ovarian carcinoma as the immunogen(Wennerberg, A. E. et al., Am. J. Pathol., 143(4), 1050-1054 (1993)).Institutional Animal Care and Use approval was obtained. Preliminary andsecondary screenings were by avidin-biotin IH (mouse IgG peroxidase kit,Vector Laboratories, Burlingame, Calif.) on tissue sections of ovariancarcinomas and selected normal adult tissues. DS6 was chosen for furtherstudies and, after several rounds of single cell cloning by limitingdilution, was isotyped as an IgG1 (ImmunoType Kit, Sigma Chemicals, St.Louis, Mo.). Supernatant was collected in batch for the studies in thisreport from DS6 cells grown in DMEM-F12 supplemented with 10% horseserum. Quantitation of murine IgG1 in the supernatant was by EIAmethodology (performed by the East Carolina University Hybridoma CoreFacility, Greenville, N.C.).

[0048] Immunohistochemistry. Tissue culture supernatant (40 ug DS6/ml)was used for IH as above with AEC (Vector Laboratories) as thechromagen. Frozen cryostat tissue sections were air-dried,acetone-permeabilized while formalin-fixed, paraffin-embedded tissuesections were de-waxed through solvents and rehydrated, prior toblocking of endogenous peroxidase with methanol/hydrogen peroxide andsubsequent immunostaining. Mouse IgG1 was used as a negative controlmab. Anti-transferrin receptor mab (IgG1) was used as a positive controlon frozen cryostat sections while anti-smooth muscle myosin mab (IgG1)was used as a positive control on formalin-fixed tissues (all controlantibodies from DAKO, Carpenteria, Calif.).

[0049] Human adult tissues. Samples of grossly normal, incidentaltissues were obtained fresh from either autopsy or the surgical benchfrom individuals without significant pathology in the primary organ ortissue; half were used for air-dried, acetone-permeabilized cryostatsections and half were fixed in buffered formalin and paraffin-embedded.Additional normal tissues as well as a pilot selection of gynecologicand related neoplasms were obtained from the archival tissue stores offormalin-fixed, paraffin-embedded tissue blocks of the Universityhospital (Table 1 and Table 2). Fresh specimens of serous ovarian tumorsand normal fallopian tubes were obtained from surgical cases andcryostat sections were cut for chloroform extraction studies. Use ofincidental human tissues had prior approval of the Institutional ReviewBoard.

[0050] Following completion of the normal tissue distribution and pilotstudy on gynecologic tumors, an expanded study was undertaken. 296formalin-fixed, paraffin-embedded human gynecologic neoplasms wereimmunohistochemically examined using DS6 as the primary antibody (Table3). A large study of non-gynecologic neoplasms followed (n=approx 1200)as seen in Tables 4-8.

[0051] Pretreated and/or preabsorbed immunohistochemical studies. Forperiodic oxidation of carbohydrates, formalin-fixed, paraffin-embeddedsections of normal human stomach, small bowel, colon, fallopian tube,breast and serous ovarian carcinoma were pretreated, prior to IH, with20 mM periodic acid in 0.5M acetate buffer, pH 5.0 as described by Cao(Cao, Y. et al., J. Histochem. & Cytochem., 45(11), 1547-1557 (1997);Cao, Y. et al., Tumor Biol. 19(1), 88-99 (1998).). Parallel controlsections were incubated in acetate buffer without periodic acid tocontrol for loss of reactivity due to pH or buffer. For detection ofsialic acid, 0.02 U/ml neuraminidase from Vibrio cholerae (reacts withsialic acids in α 2-3,-6,-8 linkages, Boehringer Mannheim/Roche,Indianapolis, Ind.), in 0.01M Ca++in PBS was used to pretreatformalin-fixed tissue sections of the above tissues with parallelcontrol sections pretreated with Ca++/PBS buffer without neuraminidase.An additional control for the adequacy of sialic acid removal (viaunmasking of PNA lectin-binding sites) was the inclusion of a section offormalin-fixed kidney, run as above, but stained with PNA-biotin (EYLabs, San Mateo, Calif.) in place of the DS6 primary and biotin-labeledsecondary antibodies. For removal of glycolipids prior to DS6immunostaining, unfixed, air-dried cryostat sections of normal fallopiantubes and serous ovarian tumors were placed in 5% chloroform in methanolfor 10 minute (Zhang, S. et al., Int. J. Cancer, 73, 50-56 (1997)).

[0052] To determine if DS6 reacts with several commercially available,highly purified carbohydrate antigens (sTn, Tn, Tf, sLea, Calbiochem, LaJolla, Calif.), the antigens were used to preabsorb DS6 prior toimmunostaining (Taylor, C. R., In: C. R. Taylor (ed.), Immunomicroscopy.A diagnostic tool for the surgical pathologist, pp.23-43, W. B.Saunders, Phildelphia (1986); Elias, J. M., Immunohistopathology: apractical approach to diagnosis, pp.53-56, American Society of ClinicalPathologists, USA (1990)). Briefly, carbohydrate antigens were added toDS6 supernatant at 10 ug, 50 ug, 250 ug and 500 ug carbohydrate to 4 ugDS6 and allowed to react at 4° C. overnight. This preabsorbedsupernatant was then used by IH to study sections of formalin-fixed,paraffin-embedded serous ovarian carcinomas and compared to controlsections stained with unabsorbed DS6 supernatant.

[0053] Double-immunolabel, two-color IH on tissue sections. Smallsections (up to 1.0 cm) of eight formalin-fixed and paraffin-embeddedserous adenocarcinomas and a metastasis of each were re-embedded intotwo paraffin blocks for single antibody and double label/sequential IH(Battifora, H., Lab. Invest., 55, 244-248 (1986)). Seven of the cases ofadenocarcinoma had their primary site in the ovary, and one case was aprimary papillary serous carcinoma of the peritoneum. A section ofnormal fallopian tube was also included in each block. A manufacturer'sprotocol (Vector Laboratories) for two-color, double-label IH was used:in brief, one of the murine mabs is used in a standard avidin-biotin IHtechnique with DAB (brown product, Vector Laboratories) as chromagen andthen the entire process is repeated on the same slide using thealternate murine mab with VIP (“Very Intense Purple”; purple product,Vector Laboratories) as the chromagen. DS6 (neat) supernatant was usedas one mab and a murine anti-Cal 25 (OCH 125 Level 1, an IgG1, SignetLaboratories, Dedham, Mass.) as the alternative mab. DAB wasconsistently used as the first chromagen and VIP as the second chromagenin the sequence. For double-label studies with both DS6 and OC125, theorder of the primary antibodies was switched while maintaining the orderof the chromagens so that each mab was evaluated both as a first andsecond mab in the sequence. Controls consisted of a primary mab (DS6 orOC125) with DAB followed by a mouse ascites negative control (SignetLaboratories) as the second mab with VIP. Antigen retrieval (10 mM, pH6.0 citrate buffer) by microwave proceeded all double-label staining toassure antigen detection by mab OC125.

[0054] Immunoblotting and SDS-PA GE gel electrophoresis. Lysates ofOVCAR5, a human ovarian carcinoma cell line (a generous gift from Dr.Thomas Hamilton, Fox Chase Cancer Center, Philadelphia, Pa.) that isreactive with DS6 by IH, were analyzed on one and two-dimensionalSDS-polyacrylamide gel electrophoresis as previously described (VanLecuwen, J. E. M. and Kearse, K. P., J. Biol. Chem., 271, 25345-25349(1996)). Gels were transferred to a nitrocellulose membrane and probedwith DS6 or control antisera diluted in PBS containing 5% powdered milk,washed, and incubated with protein A-HRP (ICN, Irvine, Calif.) in PBS(5% milk). Protein A-HRP complexes were visualized by chemiluminescenceperformed according to manufacturer's instructions (Pierce ChemicalCompany, Rockford, Ill.). N-linked glycans were removed fromprecipitated proteins using an Enzymatic Deglycosylation kit (Glyko,Inc., Novato, Calif., USA).

[0055] 2. Results

[0056] Distribution of CA6 in normal tissues and tumors by IH. Theresults of IH staining using DS6 on formalin-fixed, paraffin-embeddedtissues are similar to those obtained on acetone-permeabilized cryostatsections, consistent with an epitope that is fairly resistant to theeffects of formalin-fixation. The IH pattern of DS6 on cells of normaladult tissues is predominately an apical, epithelial cell membranelocalization in cells lining certain tubular or saccular structures(e.g., fallopian tube, pulmonary alveoli) and along the luminal-facingaspect of the inner layers of some stratified epithelia, such asurothelium (FIGS. 1, A-C). In a few instances, such as metaplasticcervical epithelium and squamous metaplasia of amnion, the entire cellmembrane can be decorated giving a rim pattern to the cell (FIG. 1, D).There is absent to minimal cytoplasmic expression of the antigen in anyof the normal human adult epithelia studied.

[0057] The immunohistochemical distribution of the CA6 antigen invarious types of normal adult tissues in given in Table 1. In normaladult tissues, the CA6 antigen is most consistently detected infallopian tube epithelium, urothelium and type 2 pneumocytes. In othertissues that have DS6 immunoreactivity, the CA6 antigen has a moreinhomogeneous and variable expression pattern. This is particularlyevident in ductal structures, where expression can vary from one ductcross section to another within a given histologic preparation.

[0058] In a pilot study of selected gynecologic neoplasms (Tables 2),CA6 is expressed along the apical membranes of many cells in ovarianserous cystadenomas. In ovarian serous adenocarcinomas, the level of CA6expression is more intense and is additionally seen in the cytoplasm ofmany malignant cells and extracellularly within gland lumen (FIG. 2, C).A similar luminal/apical pattern with variable cytoplasmic staining isalso seen in the other adenocarcinomas that are DS6 immunoreactive.Mucinous ovarian tumors are not characteristically DS6 immunoreactive asno reactivity is seen in benign and borderline mucinous tumors and isonly seen quite focally in 3 of 9 mucinous adenocarcinomas. CA6 is notdetected in normal or hyperplastic mesothelium and does not appear to becharacteristic of mesotheliomas (1/6), but is readily detected in seroussurface carcinomas of the peritoneum (3/3).

[0059] Sensitivity of CA6 epitope to periodic acid, neuraminiclase, andchloroform extraction. To characterize the CA6 epitope, sections ofDS6-reactive (IH) fallopian tube and serous ovarian carcinoma, as wellas DS6 nonreactive stomach, small bowel, colon and breast, weresubjected to a series of pretreatments with either periodic acid orneuraminidase (V. cholera) prior to immuonostaining with DS6.Pretreatment of the tissue sections with either periodic acid orneuraminidase (sialidase) prior to IH with DS6 results in completeabolishment of DS6 immunoreactivity in the sections of fallopian tubeand ovarian carcinoma and no DS6 immunoreactivity was unmasked in any ofthe sections of gastrointestinal tissues or breast. DS6 reactivityremained unaffected in the buffer control sections (FIGS. 2, A-B).Preabsorption of DS6 supernatant with commercially available purifiedcarbohydrate antigens (sTn, sLea, Tf, Tn) under conditions of antigenexcess failed to reduce the intensity of DS6 immunostaining of serousovarian carcinomas (data not shown).

[0060] Chloroform extraction studies were performed on sections offallopian tubes (2) and serous ovarian tumors (3) prior toimmunostaining with DS6. Control cryostat sections of fallopian tubesand ovarian tumors show strong staining with DS6 in the formalin-fixedas well as in the acetone-permeabilized slides. Thechloroform-pretreated slides, stained with DS6, reveal mild to moderatereduction in staining intensity in fallopian tubes and ovarian tumorsrespectively.

[0061] Immunoblotting and SDS-polyacrylamide gel electrophoresis.OVCAR5, a human ovarian carcinoma cell line reactive with DS6 by IH(data not shown) was used as a reproducible source of antigen. Anti-DS6immunoblots of OVCAR5 lysates gave a predominant band at approximately80 kilodaltons (kDa), with minor species at 75 kDa, 52 kDa, and 48 kDa(FIG. 3, B). Migration is similar under both reducing and nonreducingconditions and no reactivity with control (isotype matched) antibody isobserved (FIGS. 3, B-C). Analysis on two-dimensional nonequilibrium pHgradient electrophoresis (NEPHGE) gels indicates an isoelectric point ofapproximately 6.2-6.5 (FIG. 3, A). Digestion studies with PNGase Fresult in a reduction in molecular weight of the 80 kDa species toapproximately 75 kDa, consistent with the presence of one to two N-likedglycan chains (data not shown).

[0062] Double-immunolabel, two-color IH of tissue sections. Singleantibody and double-label, two-color IH studies were performed usingmabs DS6 and OC125 to determine if CA6 and CA125 are expressed by thesame or differing tumor cell populations. Both antibodies reacted withthe luminal aspect of fallopian tubes but only OC125 reacted with themesothelium of the fallopian tubes. All eight primary papillary serousadenocarcinomas and their metastases show moderate to strong positivityfor DS6 and OC125 with the exception of one primary tumor that has onlyscattered focal positive cells with either mab. Comparison of tumorsstained individually with either DS6 or OC125 reveal that they reactwith roughly similar proportions of tumor cells although the intensityof staining is more pronounced with DS6. While both the CA6 and CA125antigens are expressed along the luminal membranes of the neoplasticcells in many areas and in most tumors strikingly similar IH patternscan be found, there are some distinct differences. CA6 often has anadditional prominent cytoplasmic component while CA125 will occasionallyshow a distinct pattern of circumferential membrane expression on theneoplastic cells in several of the tumors, giving a honeycomb appearanceto the tumor that is not as characteristic of CA6 (FIGS. 2, C-D). Whenboth DS6 and OC125 are used in sequential, two-color IH, intenselypositive dark chromagen results from overlapping, dual localization ofchromagens in many areas of the tumors. This dark chromagen can bedifficult to distinguish from an intensely positive deposition from thefirst mab/chromagen (brown, DAB) reaction alone. However, in mosttumors, focal areas of chromagen deposition resulting from the secondmab/chromagen (purple, VIP) reaction can be clearly visualized (FIGS. 2,E-F). Such areas result from the second mab in the sequence reactingwith an antigen that is not detected by the prior application of thefirst mab.

[0063] Following the immunohistochemical studies above, a greatlyexpanded number of gynecologic neoplasms, as well as non-gynecologicneoplasms, were similarily studies for DS6 immunoreactivity. The resultsare listed in Tables 3-8. As can be seen, the CA6 antigen is notrestricted to ovarian carcinomas but can be expressed by a variety ofother carcinomas, including, but not limited to, those of the breast,endometrium, pancreas, urinary bladder and lung.

[0064] 3. Discussion

[0065] As CA6 has only a limited distribution in the normal tissuesstudied, is not detected in normal ovarian surface epithelium, yet isexpressed in serous ovarian carcinomas which arise from the surfaceepithelium (18/18), it can be described as a TAA (Suresh, M. R.,Anticancer Res., 16(4B), 2273-2277 (1996); Khawli, L. A. and Epstein, A.L., Q. J. Nucl. Med., 41, 25-35 (1997)). Immunohistochemical studies ofDS6 on a wide variety of human carcinomas, lymphomas and sarcomas can beseen in Tables 2-8 and demonstrate that DS6 is not a pan-carcinomamarker but rather is characteristic of specific types of epithelialmalignancies (e.g., characteristic of serous ovarian carcinomas butseldom reactive with colon carcinomas). Analyzing the IH distribution ofCA6 in normal tissues, similarities, as well as differences, can benoted to other reported TAAs. For instance, the pattern of reactivity inthe anterior pituitary has also been described for CA19-9 and EMA(Okubo, T. and Okabe, H., Acta Neuropathol., 93, 471-476 (1997);Pernicone, P. J. et al., In: S. S. Sternberg (ed.), Histology forpathologists, second edition, pp 1053-1074, Lippincott-Raven,Philadelphia (1997)), the staining of the intracellular canaliculus ofparietal cells is a feature of the MUC1-related mab HMFG2 (Walker, M. M.et al., J. Clin. Pathol., 48, 832-834 (1995)), while luminal staining ofductal structures is variably reported for a large number of TAAs,including the MUC1 mucin family and histo-blood group related antigens(Arklie, J. et al., Int. J. Cancer, 28, 23-29 (1981); Zhang et al.,supra (1997); Cao, Y. et al., Histochem. Cell Biol., 106, 197-207(1996)). Reports of mabs to TAAs have been numerous and, as TAAs are notunique to tumors, it is not unexpected that some similarities in theirIH distribution in normal epithelial tissues would be seen. The reporteddistribution of epithelial TAAs can also vary in the hands of differentinvestigators, perhaps reflecting differences in methodologies, gradingsystems, antibody preparation, tissue preparation, or fixation (Zhang etal., supra (1997); Stein, R. et al., Int. J. Cancer, 47,163-169 (1991);Buist, M. R. et al., J. Clin. Pathol., 48, 631-636 (1995)). We havecharacterized the IH distribution of DS6 in both cryostat andformalin-fixed, paraffin-embedded tissues to provide a range ofexperimental conditions that are in common use. Additionally, we haveclassified the types of DS6-reactive epithelial tissues as to the typesof tissues most consistently positive versus those tissues in which theexpression appears more variable. As will be discussed below, theconsistent presence or absence of CA6 in certain epithelial tissues doesappear to distinguish CA6 from certain other known TAAs. To furthercharacterize CA6, additional features of the antigen were investigated.

[0066] Pretreatment of tissue sections with either periodic acid orneuraminidase from Vibrio cholerae gives similar results. Thesetechniques are used to determine if the epitope is carbohydrate-based orsialic acid-dependent, respectively, and have also been used to unmaskhidden epitopes such as masking of the binding sites of several MUC1mabs by glycosylation (Cao et al., supra (1997); Cao et al, supra(1998)). The abolishment of DS6 immunoreactivity by both treatments isconsistent with a carbohydrate epitope that is sialic acid-dependent. NoDS6-reactive epitopes are unmasked in any of the sections by suchremoval of periodic-sensitive carbohydrates or sialic acid.

[0067] To determine if the CA6 sialoglycotope is expressed as asialoglycoprotein and/or a sialoglycolipid (ganglioside), additionalstudies were performed. SDS-PAGE immunoblots reveal that the DS6 mabdetects a predominant 80 kDa glycoprotein with N-linkages. Minor sidebands are noted that may represent proteolytic degradations of the majorband or are side reactivites of the antibody (ie, carbohydrate epitopeshared on more than one protein). No change in migration of the major 80kDa band is noted between reduced and non-reduced conditions signifyinga lack of interchain disulfide bonds (FIGS. 3, B-C). Chloroformextraction studies are used to extract lipids from tissue sections priorto DS6 immunostaining and are then compared to DS6 immunostaining ontissue sections without prior chloroform extraction. While some generalreduction in staining intensity was noted in sections of fallopian tube,a more marked reduction was noted in the sections of ovarian tumors.Whether this apparent difference in efficiency of extraction in a normaltissue (i.e., fallopian tube) as compared to tumors is quantitative orqualitative is uncertain, however, ongoing studies into the chemicalnature of the epitope may help explain the significance of this finding.The sialoglycotope nature of CA6 is similar to many of the histo-bloodgroup-related antigens such as sTn, sLea, sLex, some of which aredually-expressed as glycoproteins and glycolipids, however, unlike CA6,as glycoproteins, these are usually high molecular weight, carbohydraterich O-linked glycoproteins (mucins) (Magnani, J. L. et al., CancerRes., 43, 5489-5492 (1983); Muraro, R. et al., Cancer Res., 48,4588-4596 (1988)). The immunohistochemical distribution of thehisto-blood group related antigens, as reviewed by Zhang et al., wouldalso distinguish CA6 from the above sialyated histo-blood group antigens(Zhang et al., supra (1997)). The apical-staining pattern of DS6 on type2 pneumocytes, which lack sTn, sLea and sLex expression (Hachiya, T. etal., Virchows Arch., 434, 63-69 (1999); Atkinson, B. F. et al., CancerRes., 42, 4820-4823 (1982)), is strikingly similar to that reported forthe sTf (sialyl-Thomsen-Fridenreich) antigen (Hachiya et al., supra(1999)). However, if DS6 is reacting with the sTf epitope per se, itwould be expected to demonstrate a much broader range ofimmunoreactivity in human tissues than is seen in our studies, includingexpression in DS6-nonreactive epithelia, brain, lymphoid and othermesenchymal tissues (Cao et al., supra (1996)). The DS6 preabsorptionstudies using commercially available, highly purified sTn, sLea, Tn andTf carbohydrate antigens, in which the antigens failed to inhibit thetissue binding of DS6, also support CA6 being distinct from thesecarbohydrate antigens. Other TAAs to consider are those of approximately80 kDa such as OC133 (Berkowitz, R. et al., Am. J. Obstet. Gynecol.,146(6), 607-612 (1983); Masuho, Y. et al., Cancer Res., 44, 2813-2819(1984)), Ki-OCI-6-2 (Mettler, D. L. et al., Cancer, 65, 1525-1532(1990)), and the lactoferrin-related MAM5 (Zotter, S. et al., VirchowsArch. A. Pathol. Anat. Histopathol., 406(2), 237-251 (1985)), none ofwhich are expressed by fallopian tube, and 22-1-1, a mab to aneuraminidase resistant epitope on a 78 kDa protein, which is morecharacteristic of mucinous rather than serous ovarian tumors (Sonoda, K.et al., Cancer, 77(8), 1501-1509 (1996)).

[0068] Although the molecular weights of CA6 (80 kDa) and CA125 (>200kDa) and their distribution in normal tissues differ (CA125 has beendetected by IH in some normal epithelia of pancreas, ovary, fallopiantube, colon, gallbladder, stomach, endometrium, bronchus, lung, kidneyand in mesothelium and amnion), the IH pattern of CA6 in our series ofovarian tumors is similar to that reported for CA125 (Davis, H. M. etal., Cancer Res., 46, 6143-6148, 1986; Nouwen, E. J. et al., CancerRes., 46(2), 866-876 (1986); Dietel, M. et al., J. Cancer Res. Clin.Oncol., 111, 257-256 (1986); Itahashi, K. et al., Arch. Gyn. Obst.,243(3), 145-155 (1988)). Both antigens are more characteristic ofovarian serous carcinomas than ovarian mucinous neoplasms and both havea variegated distribution pattern, often with luminal accentuation(Mattes, M. et al., Cancer Res. (suppl.), 50, 880-884 (1990)). In IHstudies, both antigens show a spectrum of expression ranging from strongand diffuse in some tumors to patchy and variegated patterns in otherneoplasms, with strongly positive groups of cells adjacent tononreactive foci. The double-label, two-color, sequential IH studiescomparing CA6 to CA125 demonstrate that mabs DS6 and OC125, whileshowing extensive overlap, also show areas in which only one of theantibodies is expressed, providing additional, complementary coverage ofthe tumor. Such complementarity of TAA expression can have importantclinical implications, whether for tailoring mab-based cancer therapiesas “cocktails” matched to a given tumor's antigenic phenotype (Liao,S.-K. et al., Cancer Immunol. Immunother., 28, 77-86 (1989); Oldham, R.K., Mol. Biother., 3, 148-161 (1991)), or for devising panels of tumormarkers to monitor tumor progression (Cane, P. et al., GynecologicOncol., 57, 240-245 (1995)).

[0069] In the sections of fallopian tubes, CA125 is distributed alongluminal epithelial surfaces and is readily detected in the mesothelium.Conversely, CA6, which is more intensely expressed along the fallopiantube lumen, is not detected in the mesothelium. DS6 clearly recognizesan antigen that has a similar, but not identical, distribution to theCA125 antigen, with some distinct differences in patterns and sites ofantigen expression. The lack of CA6 expression in normal mesotheliumcould make DS6 a candidate for intraperitoneal antibody-targetedtherapeutic applications. TABLE 1 Immunohistochemical detection of CA6in tissue sections of normal adult tissues. Acetone-permeabilized andformalin-fixed, paraffin-embedded tissues were stained with mab DS6 asthe primary antibody by avidin-biotin immunohistochemistry. CryostatFormalin-paraffin #positive/ #positive/ Tissue #tested #tested Positivecell types/Comments Tissues with prominent, consistent CA6 expressionwithin and between specimens: Fallopian tube 3/3 6/6 Luminal/apicalepithelial cell membrane  (8/8)^(a) Lung 2/2 5/5 Apical-aspect of typeII pneumocyte; occasional epithelial cell of bronchiole Urothelium 3/38/8 Inner layers, luminal-facing aspect Tissues with low-level CA6expression, quite variable/focal within and between specimens: Pancreas2/2 5/6 Luminal-aspect of small to medium ducts Liver 1/2 3/5Luminal-aspect of occasional portal bile ducts Major salivary gland nt^(b) 2/3 Luminal-aspect of occasional medium-sized ducts Kidney 3/35/5 Luminal-aspect of scattered cells of distal nephron Endometrium,secretory 3/3 3/4 Focal luminal staining Rare surface epithelial cell,2/4 Cervix 2/2 5/6 Focal, membrane rim pattern in metaplasticepithelium, transformation zone and 1/1 ectocervical epitheliumEndocervical glands  0/2^(c) 3/6 Focal luminal staining of some glandsin transformation zone Pituitary 2/2 2/2 Focal staining of rim offollicles, anterior lobe Tissues lacking CA6 expression or rare focusonly: Cerebral cortex 0/3 0/3 Cerebellum 0/3 0/3 Spinal cord 0/3 0/3Large peripheral nerve 0/3 0/3 Trachea nt 0/2 Parathyroid 0/2 1/4 Onesmall focus of CA6 in histologically normal area of gland that elsewherehad pseudofollicle formation Thyroid 2/2 2/3 Rare apical-aspect offollicular epithelial cell Breast 1/3 2/4 Rare apical-aspect ofepithelial cell Adrenal  0/4^(d) 0/7 Esophagus 1/2 0/3 Single focus ofsuprabasal cell membrane staining, membrane rim pattern Stomach  0/4^(c)0/6 [Intracellular canaliculas of parietal cells positive, 3/4]^(c)Small intestine  0/4^(c) 0/7 Large intestine  0/4^(c) 0/6 Gallbladder nt0/3 Testes 0/4 0/5 [Weak positive rete testes, collecting duetules, 2/3;epididymis negative, 3] Prostate 0/1 0/4 [2 specimens includedperiurethral prostatic ducts, weak positive] Ovary 0/3 0/5 [oneinclusion cyst positive, numerous inclusion cysts negative] Endometrium,proliferative 0/2 0/7 Heart 0/1 0/2 Skeletal muscle 0/2 0/3 Thymus nt0/3 Spleen 0/2 0/3 Lymph node 0/2 0/3 Epidermis 0/2 0/5 Othernon-epithelial see comments Tissues consistently negative in numeroussections: mesothelium, rbc, wbc, lymphoreticular cells, fat, smoothmuscle, nerve, endothelium Placenta nt 0/5 [Focal squamous metaplasticamnion cell, rim pattern, 3/3] Chorion nt

[0070] TABLE 2 Immunohistochemical detection of CA6 in gynecologic andrelated neoplasms/hyperplasias. Formalin-fixed, paraffin-embedded tissuesections were stained with mab DS6 as the primary antibody byavidin-biotin immunohistochemistry. #positive/ Tissue #tested Ovarian:Serous cystadenoma 3/3 Serous borderline tumor 5/5 Serous adenocarcinoma11/11  (18/18)^(a) Mucinous cystadenoma 0/2 Mucinous borderline tumor0/2 Mucinous adenocarcinoma 3/9 Endometroid, borderline 0/1 Endometrial:Clear cell adenocarcinoma 2/2 Papillary serous adenocarcinoma 3/3Endometroid adenocarcinoma 1/1 Benign polyp 0/1 Peritoneum: Mesothelioma1/6 Reactive mesothelial hyperplasia 0/1 Serous surface carcinoma ofperitoneum 2/2 (3/3)

[0071] TABLE 3 Expression of Tumor-Associated Antigen CA6 inGynecological Neoplasms as Immunohistochemically Detected by MonoclonalAntibody DS6 1 + 2 + 3 + Reactive Nonreactive (Cases with (Cases with(Cases with > Cases/ (Cases with < 1-9% 10-49% 50% Total 1% neoplasticneoplastic neoplastic Major Number neoplastic cells cells cells Patternsof Neoplasm of Cases cells reactive) reactive) reactive) reactive)Reactivity^(I) Uterine Cervix Squamous Cell Carcinoma 10/14 4 4 3 3 Me,Cy Squamous Dysplasia 0/7 7 Endocervical 0/2 2 Adenocarcinoma in SituEndocervical 2/3 1 1 1 Lu, Me Adenocarcinoma Adenosquamous Carcinoma 2/22 Me, Cy Endometrium Papillary Serous 6/6 2 2 2 Lu, Me AdenocarcinomaEndometriod 14/16 2 7 4 3 Lu, Me Adenocarcinoma Clear Cell Carcinoma 8/10 2 1 7 Lu, Me, Cy Mucinous Adenocarcinoma 3/5 2 1 2 Me, CyMalignant Mixed Mullerian 10/18 8 5 1 4 Me, Cy Tumor, carcinomatouscomponent Choriocarcinoma 0/1 1 Molar Pregnancy 0/3 3 Uterus Low GradeEndometrial 0/3 3 Stroma Sarcoma Undifferentiated 0/1 1 EndometrialSarcoma Leiomyoma 0/7 7 Leimyosarcoma 0/6 6 Ovary Serous Cystadenoma 9/12 3 1 8 Lu Papillary Serous Borderline 10/10 1 2 7 Lu TumorPapillary Seroris 56/58 2 4 9 43 Lu, Me, Cy Adenocarcinoma PapillarySerous Carcinoma 14/14 1 13 Lu, Me, Cy Metastases Mucinous Cystadenoma 0/10 10 Mucinous Borderline 0/8 8 Tumor, Intestinal Mucinous Borderline2/2 1 1 Lu Tumor, Mullerian Mucinous Adenocarcinoma  0/10 10 Clear CellCarcinoma 1/8 7 1 Me Endometriod 5/8 3 3 1 1 Lu, Me AdenocarcinomaBenign Brenner Tumor 6/9 3 2 1 3 Me, Lu, Cy Borderline Brenner Tumor 1/11 Me, Cy Granulosa Cell Tumor  0/15 15 Fibroma/Thecoma  0/11 11 SertoliCell Tumor 0/1 1 Yolk Sac Tumor 0/2 2 Struma Ovarii 0/1 1 Dysgerminoma0/3 2 Vulva Squamous Cell Carcinoma 2/3 1 2 Me, Cy HidradenomaPapilliferum 2/2 2 Me Peritoneum Papillary Serous Surface 2/2 1 1 Lu, MeCarcinoma Malignant Mesothelioma of 0/2 2 peritoneum Endosalpingiosis1/1 1 Lu (nonneoplastic) Mesothelium (incidental 0/7 7 nonneoplastic)

[0072] TABLE 4 Expression of Tumor-Associated Antigen CA6 in EpithelialNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS6Reactive Nonreactive 1 + 2 + 3 + Cases/ (Cases with < (Cases with (Caseswith (Cases with > Major Total 1% 1-9% 10-49% 50% Patterns of Numbercells cells cells cells CA6 Neoplasm of Cases reactive) reactive)reactive) reactive) Expression Breast Infiltrating Ductal Carcinoma24/29 5 3 8 13 Lu, Me, ICL Infiltrating Lobular  5/11 6 1 1 3 ICLCarcinoma Pleomorphic Lobular 1/2 1 1 ICL, Lu Carcinoma ColloidCarcinoma 6/7 1 3 3 Lu, Me Invasive Apocrine 1/1 1 ICL CarcinomaMedullary Carcinoma 1/1 I Lu, Me, ICL Tubular Carcinoma 1/1 1 Lu, ICLInvasive Papillary 1/1 1 Lu, Me Carcinoma Brain Meningioma  3/25 22 2 1Cy, Me Craniopharyngioma 1/2 1 1 Lu Gallbladder GallbladderAdenocarcinoma 3/7 4 1 2 Lu, Me Cystic Duct Adenocarcinoma 1/2 1 1 LuGastrointestinal Colonic Adenocarcinoma  3/21 18 2 1 Lu, Me MetastaticColon 0/3 3 Adenocarcinoma Mucinous Colon 0/6 6 Adenocarcinoma ColonicTubular Adenoma 0/1 1 Cloacogenic Carcinoma 1/3 2 1 Lu, Me DuodenalAdenocarcinoma 0/1 1 Esophageal Adenocarcinoma 1/4 3 1 Lu EsophagealSquamous 2/9 7 1 1 Me, Cy Carcinoma Gastric Adenocarcinoma  7/19 12 2 5Lu, Me Gastric Squamous 1/1 1 Me, Cy Kidney Clear Cell Carcinoma 13/18 513 Me, Lu Papillary Carcinoma 3/5 2 1 1 1 Lu, Me Granular Carcinoma 5/83 2 3 Me Chromophobe Carcinoma 3/4 1 2 1 Lu, Me, Cy Oncocytoma 2/2 1 Lu,Me, Cy Cortical Adenoma 1/1 1 Lu, Me Wilms Tumor 1/9 8 1 Lu in tubulesLarynx Squamous Cell Carcinoma 10/27 17 8 1 1 Me, Cy Squamous Papilloma1/1 1 Lu, Me Liver Hepatocellular Carcinoma 1/8 7 1 Lu, Me, Cy LungAdenocarcinoma  8/12 4 4 1 3 Lu, Me Squamous Cell Carcinoma  6/15 9 1 41 Me, Cy Adenosquamous Carcinoma 1/1 1 Me Pancreas Adenocarcinoma 8/9 12 3 3 Lu, Me Pleura Malignant Mesothelioma 0/6 6 Me Prostate AcinarAdenocarcinoma  4/13 9 3 1 Lu, Me Prostatic Duct 0/5 5 AdenocarcinomaSalivary Gland Epithelial Neoplasms [See 21/90 69 12 6 3 Fig.] SkinEpithelial Neoplasms [See 19/79 60 15 4 Fig.] Testicle Teratoma,epithelial elements 2/5 3 2 Lu Thyroid Follicular Adenoma 0/2 2Follicular Carcinoma  1/17 16 1 Lu Hurthle Cell Adenoma 2/5 3 1 1 LuHurthle Cell Carcinoma 2/5 3 1 1 Lu Hyperplastic Hurthle Cell 1/1 1 LuNodule Insular Carcinoma 1/1 1 Lu, Me Papillary Carcinoma 16/27 11 9 7Lu, Me Thymus Thymoma  0/17 17 Lu, Me Thymic Carcinoma 0/1 1 SquamousCell Carcinoma 1/1 1 Lu, Me Urinary Bladder Transistional Cell Carcinoma18/21 3 8 5 5 Lu, Me Squamous Cell Carcinoma 0/1 1

[0073] TABLE 5 Expression of Tumor-Associated Antigen CA6 in SkinNeoplasms as Immunohistochemically Detected by Monoclonal Antibody DS6Reactive 1 + 2 + 3 + Cases/ Nonreactive (Cases with (Cases with (Caseswith > Major Total (Cases with < 1-9% 10-49% 50% Patterns of Number 1%cells cells cells CA6 Neoplasm of Cases cells reactive) reactive)reactive) reactive) Expression Basal Cell Carcinoma  0/19 19 ChondroidSyringoma 0/1 1 Cylindroma 0/3 3 Dermatofibroma 0/9 9 Dermatofibosarcoma0/3 3 Protuberans Eccrine Aerospiroma 11/13 2 10 1 Me, Lu EccrineSpiradenoma 1/7 6 1 Lu Ecerine Carcinoma 0/1 1 Hemangioma 0/8 8Leiomyoma 0/8 8 Leiomyosarcoma 0/1 Merkel Cell Carcinoma 1/2 1 PunctateCy, Me Neurofibroma  0/13 13 Nevus Sebaceous 0/5 5 Pilomatricoma 0/1 1Poroma 3/6 3 2 1 Me, Lu Seborrheic Keratosis 0/1 1 Spiradenoma 1/7 6 1Lu Syringoma 0/2 2 Syringocystadenoma 3/3 1 2 Lu PapilliferumTrichepithelioma  0/10 10 Urticaria Pigmentosa 0/3 3 Xanthogranuloma 0/44 Angiofibroma 0/2 2 Angiosarcoma 0/2 2 Glomangioma 0/1 1Hemangiopericytome 0/2 2 Malignant 0/1 1 Hemangiopericytoma Blue Nevus0/5 5 Spitz Nevus 0/2 2 Malignant Melanoma  0/10 10

[0074] TABLE 6 Expression of Tumor-Associated Antigen CA6 in SalivaryGland Neoplasms as Immunohistochemically Detected by Monoclonal AntibodyDS6 Reactive 1 + 2 + 3 + Cases/ Nonreactive (Cases with (Cases with(Cases with > Major Total (Cases with < 1-9% 10-49% 50% Patterns ofNumber 1% cells cells cells CA6 Neoplasm of Cases cells reactive)reactive) reactive) reactive) Expression Acinic Cell Carcinoma 0/8 8Adenoid Cystic Carcinoma  7/14 7 5 1 1 Lu Basal Cell Adenoma 1/4 3 1 LuBasal Cell Carcinoma 0/1 1 Clear Cell Carcinoma 0/5 5 CanalicularAdenoma 0/1 1 Malignant Mixed Tumor 0/1 1 Mucoepidermoid Carcinoma 3/4 13 Lu, Me Pleomorphic Adenoma  4/30 26 4 Lu Polymorphous Low Grade 0/1 1Carcinoma Salivary Duct Carcinoma 4/5 1 2 2 Lu, Me, Cy Warthin Tumor 2/16 14 2 Lu

[0075] TABLE 7 Non-Epithelial Neoplasms Which Do Not ExpressTumor-Associated Antigen CA6 as Studied Immunohistochemically withMonoclonal Antibody DS6 TOTAL CASES SITE NEOPLASM STUDIED AdrenalNeuroblastoma & 21 Ganglioneuroblastoma Pheochromocytoma 4 BreastFibroadenoma 1 Brain Glioblastoma 40 Multiforme Anaplastic 5 AstrocytomaOligodendroglioma 9 Pilocytic 12 Astrocytoma Medulloblastoma 10Ependymoma 15 Ganglion Cell 4 Tumors Central 1 Neurocytoma Schwannoma 29Hemanioblastoma 6 Gastro- Appendiceal 5 intestinal Carcinoid TumorAppendiceal 1 Adenocarcinoid Colonic Carcinoid 5 Tumor Gastric Carcinoid6 GI Mesenchymal 17 Tumors Lung Carcinoid Tumor 21 Small Cell 5Carcinoma Large Cell 6 Neuroendocrine Carcinoma Lymphoma Diffuse SmallCell 31 Lymphomas Large Cell 19 Lymphomas Follicular 36 Lmphomas Blastic11 Lymphomas Hodgkins Disease 29 Multiple Myeloma 23 Langerhans Cell 8Histiocytosis Leukemias Acute 4 Lymphoblastic Leukemia Acute 5Myelogenous Leukemia Chronic 5 Myelogenous Leukemia Oral Ameloblastoma 6Tissues Pancreas Islet Cell Tumor 6 Parathyroid Adenoma 8 Skin BlueNevus 5 Spitz Nevus 2 Skin Malignant 10 Melanoma Dermatofibroma 9Dermatofibosarcoma 4 Protuberans Hemangioma 8 Leiomyoma 8 Leiomyosarcoma1 Neurofibroma 13 Urticaria 3 Pigmentosa Xanthogranuloma 4 Angiofibroma2 Angiosarcoma 2 Glomangioma 1 Hemangiopericytoma 2 Malignant 1Hemangiopericytoma Soft Angiosarcoma 1 Tissue Alveolar Soft 1 PartsSarcoma Chondrosarcoma 2 Fibrosarcoma 2 Granular Cell 7 TumorHemangiopericytoma 1 Leiomyosarcoma 14 Liposarcoma 4 Malignant 6 FibrousHistiocytoma Malignant 6 Schwannoma Neurofibroma 1 Osteosarcoma 2Rhabdomyosarcoma 3 Undifferentiated 2 Sarcoma Paraganglioma 5 Primitive3 Neuroectodermal Tumor Testicle Choriocarcinoma 4 Embryonal 16Carcinoma Seminoma 18 Teratoma, Non- 5 epithelial elements Yolk SacTumor 12 Thyroid Medullary 3 Carcinoma

[0076] TABLE 8 Non-Epithelial Neoplasms Which Express Tumor-AssociatedAntigen CA6 as Detected Immunohistochemically by Monoclonal Antibody DS6Reactive 1 + 2 + 3 + Cases/ Nonreactive (Cases with (Cases with (Caseswith > Total (Cases with < 1-9% 10-49% 50% Major Patterns Number 1%cells cells cells of CA6 Neoplasm of Cases cells reactive) reactive)reactive) reactive) Expression Merkel Cell Carcinoma 1/2 1 1 Membranous& cytoplasmic Pituitary Adenoma  2/21 19 2 Luminal in pseudorosettes;punctate in cytoplasm Small Intestinal Carcinoid 1/9 8 1 Luminal inTumor rosettes Synovial Sarcoma 1/3 2 1 Luminal and membranous

[0077] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A monoclonal antibody selected from thegroup consisting of monoclonal antibody DS6, monoclonal antibodies thatspecifically bind to the epitope bound by monoclonal antibody DS6, andfragments of the foregoing that specifically bind to the epitope boundby monoclonal antibody DS6.
 2. A monoclonal antibody according to claim1 coupled to a detectable group.
 3. A monoclonal antibody according toclaim 1 coupled to a detectable group, said detectable group selectedfrom the group consisting of radiolabels, enzyme labels, fluorescentlabels, metal atoms, and chemiluminescent labels.
 4. A monoclonalantibody according to claim 1 coupled to a solid support.
 5. Amonoclonal antibody according to claim 1 coupled to a solid Support,said solid support selected from the group consisting of beads, plates,slides and microtiter plate wells.
 6. A monoclonal antibody according toclaim 1 coupled to a therapeutic agent.
 7. A monoclonal antibodyaccording to claim 1 coupled to a therapeutic agent, said therapeuticagent selected from the group consisting of radioisotopes, cytotoxicagents, chemotherapeutic agents, metals, and chemiluminescent agents. 8.A method of treating cancer in a subject in need thereof, comprising,administering to a subject afflicted with cancer a monoclonal antibodyin a therapeutically effective amount; said monoclonal antibody selectedfrom the group consisting of monoclonal antibody DS6 and monoclonalantibodies that specifically bind to the epitope bound by monoclonalantibody DS6, and fragments of the foregoing that specifically bind tothe epitope bound by monoclonal antibody DS6.
 9. A method according toclaim 8, wherein said cancer is selected from the group consisting ofovarian, endometrial, urinary bladder, pancreas, breast, and lungcancer.
 10. A method according to claim 8, wherein said cancer is serousadenocarcinoma of the ovary.
 11. A method according to claim 8, whereinsaid antibody is coupled to a therapeutic agent.
 12. A method accordingto claim 8, wherein said antibody is coupled to a therapeutic agentselected from the group consisting of radioisotopes, cytotoxic agents,chemotherapeutic agents, metals, and chemiluminescent agents.
 13. Amethod according to claim 8, wherein said antibody is coupled to aradioisotope.
 14. Isolated tumor-associated antigen CA6, an about 80 kDaN-linked glycoprotein, reduced or non-reduced, with a Pi of about6.2-6.5, and containing a sialiadase and periodate sensistive epitopecalled DS6.
 15. The isolated tumor-associated antigen CA6 which isspecifically bound by monoclonal antibody DS6.